1. Field of the Invention
This invention generally relates to the field of stem cell development, and in particular to the use of chemical agents to prepare populations of specific neurons, for example, dopaminergic neurons, and the applications of those neurons.
2. Related Art
Pluripotent stems cells, including embryonic stem (ES) cells and induced pluripotent stem cells, hold great promise for studying early development and for use in cell therapy. The same is true of adult and embryonic neural stem cells. Because such cells can proliferate in culture and maintain their potential for differentiating into different cell types, they can provide an almost unlimited supply of cells for treating a variety of diseases. A particularly active area of research is the treatment of nervous system diseases using cell therapy. One approach to the treatment of degenerative nervous system diseases is to transplant specific neurons, such as dopaminergic neurons, into affected areas of the nervous system.
Potential sources of those neural-lineage cells are cultures of specific neurons prepared by differentiating ES cells, induced pluripotent stem (iPS) cells and other types of stem cells in vitro. Methods of preparing primate ES cell cultures have been described for human, rhesus monkey, and marmoset ES cells (U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913). The methods involve removing the trophoectoderm layers from blastocysts, then plating the remaining inner cell mass cells onto a feeder layer of gamma-irradiated mouse embryonic fibroblasts. After 7-21 days in culture, cell outgrowths are removed, dissociated, then replated onto embryonic feeder layers. Colonies that form are then picked, briefly trypsinized to dissociate, then replated on embryonic feeder layers. The cells can be routinely split every 1-2 weeks using brief trypsinization.
Unfortunately, although a heterogeneous mixture of different cell types derived from pluripotent stem cells is easy to obtain in culture, their targeted differentiation towards a specific lineage remains challenging. In general, spontaneous differentiation of ES cells in culture produces a heterogeneous mixture of cells, only some of which may be neural cells. Thus, spontaneous differentiation is not an effective means of providing specific neurons.
Small molecules have been tested for their ability to influence ES cell differentiation. For example, retinoic acid has been used to induce neuronal differentiation of ES cells. However, exposure to retinoic acid leads to differentiation mainly of glial cells, while human ES cells exposed to retinoic acid differentiate towards epithelial cells (Metallo et al., 2007). Generation of dopaminergic neurons from mouse embryonic stem cells (CGR8 line) is a promising tool for Parkinson's disease as a source for future cell therapy. Many protocols of dopaminergic differentiation of mouse embryonic stem cells (CGR8 line) are currently available. However, most procedures include generation of heterogeneous cell population that not sufficiently pure and efficient. Thus, additional compounds and alternative methods of neural differentiation from human ES cells and other types of stem cells are needed to exploit the uses of cell therapy for treatment of neurological diseases and neurotoxicity testing.